I. Field of the Invention
The present invention relates generally to the use of viruses as biological therapeutics for treating diseases, disorders or conditions associated with cellular proliferation, and in particular, for treating malignant conditions such as cancer. More specifically, invention embodiments as disclosed herein relate to novel oncolytic attenuated reoviruses.
II. Description of the Related Art
Cancer includes a broad range of diseases characterized by the presence of inappropriate or unregulated cellular proliferation in a variety of cells and tissues. Worldwide, approximately one in four humans is afflicted with one of the various forms that cancer may take, and reliable therapeutic strategies remain a major clinical challenge for most cancer types. Current approaches include surgical excision of cancerous tissues containing malignant tumors, and radiological, chemotherapeutic or immunotherapeutic ablation of neoplastic or malignant cells. Each of these approaches provides less than ideal efficacy, with shortcomings that include incomplete removal of cancer cells and/or undesired damage or toxicity to normal, healthy tissues and/or inadequate delivery of the anti-cancer agent to the malignantly transformed target cells.
Oncolytic properties of reoviruses have been recognized in the past (Bennette et al., 1967), and more recently, naturally occurring human reoviruses have received attention as candidate therapeutic agents for certain types of cancer (Chiocca, 2002; Everts et al., 2005). Specifically, certain human reoviruses exhibit oncolytic activity, or the ability to preferentially and productively infect, and induce lysis of, cancer cells in which one or more of various altered oncogenic pathways are present. In one example, naturally occurring reoviruses are oncolytic when contacted with activated Ras oncogene-dependent tumor cells. Such oncolysis of activated oncogene-associated cancer cells proceeds through a mechanism that involves Ras pathway-mediated impairment of phosphorylation of double-stranded RNA-activated protein kinase (PKR), which is consequently unable to phosphorylate the translation initiation factor eIF-2α, thereby creating permissive conditions for translation of reoviral gene transcripts. (Coffey et al., 1998; Chiocca, 2002; see also U.S. Pat. No. 6,261,555; US Pat. Pub. US 2005/0063954). Reoviruses also exhibit oncolytic potential in myc-overexpressing lymphoid malignancies (Alain et al., 2002).
The reoviruses (Reoviridae) comprise a family of naturally occurring, non-enveloped viruses having a double-stranded RNA (dsRNA) genome that is divided into ten segments and enclosed by two concentric icosahedral protein capsids. Infectious mammalian reovirus virions of various tropisms occur as particles of approximately 85 nm in diameter. The virion outer capsid includes several distinct protein species, among them σ-1 (σ1, 50 kDa) which mediates viral attachment to host cell surfaces (Lee et al., 1981; Duncan et al., 1991; Nataga et al., 1987; Turner et al., 1992) via discrete carbohydrate-binding (Chappell et al., 1997; Chappell et al., 2000; Connolly et al., 2001) and virion-anchoring (Mah et al., 1990; Fernandes et al., 1994; Lee et al., 1994) domains. σ1 is a product of bicistronic reoviral S1 gene, which also encodes a non-structural protein designated σ1s using a distinct but overlapping reading frame (Ernst et al., 1985; Jacobs et al., 1985; Sarkar et al., 1985). Reoviral particles that lack σ1 have been reported to be non-infectious (Larson et al., 1994). The reoviral S1 gene is believed to play a significant role in determining reoviral pathogenesis (Haller et al., 1995; Wilson et al., 1994; Kaye et al., 1986; Weiner et al., 1980).
The other major reovirus outer capsid proteins, σ3 (encoded by the reoviral S4 gene, e.g., Ahmed et al., 1982; Giantini et al., 1984) and μ1 (encoded by the reoviral M2 gene, e.g., Wiener et al., 1988; Hooper et al., 1996), are present along with σ1 in intact reovirus virions, but following exposure of the virion to certain proteolytic conditions an altered structure known as in intermediate or infectious subvirion particle (ISVP) results, in which σ1 persists but σ3 is lost and two defined μ1 cleavage products remain (Dryden et al., 1993; Jane-Valbuena et al., 1999; Chandran et al., 1999; Chandran et al., 2001). ISVPs may thus result from expose of intact reovirus virions to proteolytic environments such as those found intracellularly within late endosomes or lysosomes following reoviral host cell infection (via cell surface binding and internalization), or as may be encountered via a natural enteric route, or by artificial means. Following ISVP penetration of the endosomal (or lysosomal) membrane to gain access to the infected host cell's cytoplasm, σ1 and μ1 proteins are lost to yield a reovirus-derived particle known as a core particle, which is capable of transcribing its viral mRNA contents but which, unlike virions and ISVPs, is no longer infectious.
Initially identified as an apparently innocuous infectious pathogen in the human respiratory and gastrointestinal tracts, the human reovirus has long been recognized for its striking cytocidal activity upon infection of certain types of transformed cells (Duncan et al., 1978; Hashiro et al., 1977). More recently, the relationship between tumor cells containing an activated Ras oncogene and susceptibility of such cells to reoviral oncolysis has been established (Coffey et al., 1998; Strong et al., 1998). Subsequent demonstration of reovirus role in inducing cancer cell apoptosis suggested at least one mechanism by which reoviral oncolysis proceeds (Clarke et al., 2001), and considerable efforts have been undertaken to develop cancer therapeutics using naturally occurring reoviruses (e.g., U.S. Pat. Nos. 6,565,831; 6,811,775; 6,455,038; 6,808,916; 6,528,305; 6,703,232; 6,136,307; 6,344,195; 6,110,461; 6,261,555; 6,576,234; U.S. Patent Pub. Nos. US2005/0063954; US 2005/0026289; US2004/0146491; US2002/0168344; US2004/0126869; US2004/0265271; US2005/0019308).
However, despite reoviral tropism for, and lysis of, Ras-activated tumor cells, efforts to use reoviruses as therapeutic oncolytic agents have been hampered by a number of factors, including (i) as a dsRNA virus having a segmented genome, reovirus is not readily amenable to refinements by genetic engineering (Russell, 2002; Brown et al., 2001); (ii) among transformed cells, reovirus is believed to productively infect only those cells having an activated ras pathway, which accounts for about 30% of human cancers; (iii) many in vivo protocols for reoviral oncolysis employ immunosuppressed or immunocompromised hosts and such fail to consider the effects of anti-reoviral immune responses or of a generally immunosuppressed state (Everts et al., 2005); (iv) reoviral tropism is not strictly limited to cancer cells and naturally occurring reoviruses may not be clinically innocuous, with animal models revealing reoviral infection of cardiac myocytes and endothelial cells (Loken et al., 2004) and reoviral induction of undesirable phenomena such as hemorrhage, fibrosis, hepatitis, lymphoma, pancreatitis, necrotizing encephalitis and myocarditis (Loken et al., 2004, and references 23-27 therein); and (v) as with other oncolytic regimes, oncolytic reoviral treatments may also compromise the integrity of the host stem cell compartment. Wild-type reovirus is known, for instance, to adversely affect development of rat and murine embryos, retarding development and inhibiting blastocytst formation (Priscott, 1983; Heggie et al., 1979).
Clearly there is a need in the art for improved reovirus compositions and methods that more selectively and efficiently mediate oncolysis. The present invention addresses such needs and provides other related advantages.